Molecular characterization of genes involved in chitin metabolism during the molt cycle in morotoge shrimp (Pandalopsis japonica, Balss, 1914)

Abstract

Abstract

Chitin is long unbranched insoluble polysaccharides of an amino sugar N-acetyl-β-D-glucosamine linked together by β-1,4-glycosidic linkage. It is one of the most abundant polysaccharides in natures found as structural constituents of fungal cell wall, the cuticle and the integuments of some animals particularly arthropods. In crustaceans, chitin is a major component of exoskeleton and made a external covering of all over the body, it must be shed off periodically and regenerate the new one in each and every molting cycles. Chitin polymer is synthesized by a membrane-bound enzyme, chitin synthase and several chitinolytic enzymes are responsible for depolymerization of chitin. Among them, chitinase is the endoglycosidases that cleave the N-acetyl glucosamine residue in chitin which is further degraded by N-acetyl-β-glucosaminidase for the production of monomer of N-acetyl-β-glucosamine. Since crustacean grows by periodic ecdysis (shedding of old exoskeleton) and subsequent reconstruction of a new one. Therefore, chitin synthesis by chitin synthase and degradation by chitinases play a pivotal role in the crustacean physiology for proper development and growth. In this experiment, five hepatopancreatic and one epidermal chitinases have been isolated from the pandalid shrimp, Pandalopsis japonica by using polymerase chain reaction (PCR), cloning method and bioinformatic analysis of expressed sequence tags (ESTs). The cDNAs, designated PjCht1, 2, 3A, 3B, 3C, and 4, encoded proteins ranging from 388 to 607 amino acid residues in length (43.61 – 67.62 kDa) and displayed a common structural organization: an N-terminal catalytic domain, a Thr/Pro-rich linker region, and either 0 (PjCht2, 3A), 1 (PjCht1, 3B, and 3C), or 2 (PjCht4) C-terminal chitin-binding domain(s) (CBD). Six chitinases have been classified into at least three groups. Group 1 chitinase, PjCht-1 was most closely related to insect group 1 chitinase and may have function in the digestion of peritrophic membrane. Group 3 chitinases, designated by PjCht3A, 3B, and 3C, may have function in digestion of chitin-containing food and defense against pathogens. Finally, PjCht-4 named as group 4 chitinase have two chitin binding domains (CBDs) and their function is unknown. All of the five isolated chitinases were expressed in the hepatopancreas and intestine, have been termed as hepatopancreatic chitinase on the basis of expression profile. Eyestalk ablation (ESA) down-regulated the hepatopancreatic chitinase expression (PjCht1, 3A, and 3C); PjCht3B expression was not significantly affected by ESA. Among crustacean, we have isolated two catalytic domains containing chitinase (PjCht2) for the first time. We failed to isolate the 5’prime UTR from the sequence and difficult to generalize the number of catalytic domain in the genes. Multiple catalytic domain containing chitinases, PjCht2 was expressed in the epidermis and SG/X-organ complex. Tissue distribution profile indicates that the PjCht2 is epidermis specific chitinase and mRNA transcripts were increased in response to increased ecdysteroids level after ESA and administration of 20-hydroxy ecdysone (20E). In order to understand the functional studies of PjCht2, siRNAs were synthesized from the corresponding region of the target gene which showed down regulation of mRNA transcripts in cuticular tissues. Injection of gene specific siRNA duplexes silenced the expression of PjCht2 resulted reduced molting frequency or death. This findings suggest that the epidermal gene, PjCht2, may have role in molting. Chitin formation is catalyzed by the chitin synthase (UDP-N-acetyl-D-glucosamine: chitin 4-B-N-acetylglucosaminyltransferase; EC 2.4.1.16), which belongs to the family 2 of glycosyltransferases (GTF2) and utilizes UDP-N-acetylglucosamine as activated sugar donor to form chitin polymer . We isolated the full cDNA encoding chitin synthase by conventional cloning strategy which encoded 1525 amino acids containing transmembrane protein. Molecular weight of putative protein is 175kDa and has been divided into three domains. N-terminal (domain A) and C-terminal domain (domain C) contain 9 and 7 transmembrane helices (TMH) respectively whereas catalytic domain (domain B) has two signatory motifs, EDR and QRRRW. Moreover, it has 2 coiled-coil domains in the C-terminal region which is used for protein-protein interaction. Phylogenetic analysis was performed to understand the evolutional relationship with known crustacean species, insects, yeast and nematode which indicates that PajCHS is closely related with insect CHS1 group gene. The tissue distribution of the PajCHS transcript was determined by end-point RT-PCR and subsequent agarose-gel electrophoresis of PCR products. Tissue expression profile indicated that epidermis, hepatopancreas, intestine and gill were the major sites for PajCHS transcript. Little expressions was also identified from sinus gland/X-organ complex (SG/XO). qPCR was performed to determine the effects of molt induction by ESA on the expression of PajCHS. mRNA levels of PajCHS in epidermis and intestine were significantly increased at 7th day comparing to control. Injection of 20E also upregulated the mRNA transcripts after the 7th day in epidermis similarly, in gill it was drastically increased after 12hours. These results indicate that mRNA transcripts were regulated by the increased level of ecdysteroids. Finally, the identified chitinase and chitin synthase will help us to understand the complex process of chitin metabolism in crustacean and it would also be excellent biomarker for ecotoxicological study.